The overall objective of the studies proposed in this competing continuation application remains the elucidation of the biochemical mechanisms whereby cells are lethally injured. Having addressed previously (1992-1997) the mechanisms of liver cell death following an inhibition of mitochondrial electron transport, as a consequence most importantly of ischemia anoxia, over the last three years we have studied five models of apoptosis. The major conclusion that we reached is that the cell death in necrosis as well as in each model of apoptosis studied is a consequence of induction of the mitochondrial permeability transition (MPT). In the models of apoptosis considered, our data implicate the proteins Bax or Bid in induction of the MPT. Thus, the work proposed in the present application is based on the hypothesis that the MPT is a central event in the cell death from either necrosis or apoptosis. Furthermore, we propose that these two killing processes are distinguished in at least two ways. First, necrosis and apoptosis differ with respect to the mechanism of induction of the MPT. Second, apoptosis is characterized by the energy-dependent activation of caspases as a consequence of the MPT. Such a role for caspases cannot occur with necrosis where energy depletion is a necessary event in the induction of the MPT, a situation most clearly evidenced by ischemia/anoxia. The specific aims of the present proposal are to pursue the implications of this initial reconstruction of the mechanisms mediating both necrosis and apoptosis. Aim #1 - What is the mechanism of Bax activation in apoptosis, a process that results in induction of the MPT? In those cells that contain both Bax and Bid (L929 fibroblasts and HeLa cells), we propose to distinguish between two hypotheses. Bax and Bid are redundant proteins and act independently. Bax activation (and that of Bid) is the result of a conformational change induced by alterations in cytosolic pH and/or ion composition. Alternatively, Bax translocation to the mitochondria is secondary to a primary activation of Bid. The mechanism action is also considered of other pro- and antiapoptotic proteins, including ERK (antiapoptotic) and JNK (proapoptotic) classes of MAP kinases, as well as NF- (antiapoptotic). Aim #2- What is the energy-independent mechanism coupling the MPT to the loss of cell viability? With both necrosis and apoptosis, lethal cell injury depends on the MPT. We propose to test the hypothesis that in both necrosis and apoptosis direct physical connections through the cytoskeleton between the outer mitochondrial membrane (porin in particular) and the plasma membrane mediate the energy-independent coupling of the MPT to the loss of viability represented by lethal plasma membrane injury. Aim #3- How in apoptosis does the energy-dependent activation of caspases by cytochrome c as a consequence of the MPT alter (replace or modify) the mechanism of cell killing? It is our hypothesis that the phenotypic features distinguishing apoptosis from necrosis occur in this phase and are largely mediated by caspases. It is our aim to detail the specific biochemical mediators of the major phenotypic manifestations of both necrotic and apoptotic cell death.